1. Field of the Invention
Escherichia coli 0157:H7, also known as enterohemorrhagic E. coli (EHEC), has been associated with recent outbreaks of foodborne diseases. Sporadic cases of hemorrhagic colitis and hemolytic uremic syndrome have occurred worldwide. These outbreaks have been attributed to the presence of the pathogenic microorganism in ground beef since beef and dairy cattle are known to carry the organism in their intestinal tracts and carcasses are frequently contaminated during the slaughter process. The most visible outbreak in the United States occurred in the Western states and involved over 500 cases and several deaths of young children. Investigations by the Centers for Disease Control indicate that EHEC is the third most important cause of foodborne illness in the U.S., and the incidence of the disease is increasing. There is thus a strong incentive to develop a quick and sensitive assay method for the detection of the microorganism in beef products such as ground beef, on beef carcasses during inspection and in cattle fecal specimens. This invention relates to novel primers which can be used to detect pathogenic E. coli by specifically amplifying a fragment of a plasmid found in all strains tested by polymerase chain reaction (PCR).
2. Background of the Invention
EHEC has emerged as a foodborne pathogen of considerable public health importance. Present detection methodologies for this pathogen in foods, however, are either expensive, time-consuming cumbersome, have low specificity and sensitivity or require extensive training to perform. Since most methods require prior enrichment steps, the amount of time needed to obtain final confirmatory results is prolonged.
Virtually all EHEC harbor a large .about.60-MDa plasmid (Fratamico et al. 1993. J. Med. Microbiol. vol. 39, pp. 371-381). It is currently unknown whether the presence of the plasmid plays a role in virulence. Known virulence factors include the production of one or more types of bacteriophage-encoded Shiga-like toxins, SLTs or verotoxins (Strockbine et al. 1986. Infect. Immun. vol. 53, pp. 135-140; Karmali, M. A. 1989. Clin. Microbiol. Rev. vol. 2, pp. 15-38), and the ability of the bacteria to intimately adhere to the intestinal mucosa by an attaching and effacing mechanism (Tzipori et al. 1989. Infect. Immun. vol. 57, pp. 1142-1150).
It has been suggested that the protein product of the EHEC eaeA gene may be necessary for attaching and effacing adhesion (Yu and Kaper. 1992. Mol. Microbiol. vol. 6, pp. 411-417). The gene has been cloned and sequenced and the product determined to be a 97-kDa outer membrane protein, called intimin.sub.0157 (Louie et al. 1993. Infect. Immun. vol. 61, pp. 4085-4092). The genes encoding SLT-I and SLT-II have also been cloned and sequenced (Jackson et al. 1987. FEMS Microbiol. Lett. vol. 44, pp. 109-114), and immunological and DNA-based methods such as DNA hybridization have been developed for the detection of SLT-producing E. coli (Gannon et al. 1992. Appl. Environ. Microbiol. vol. 58, pp. 3809-3815; Paton et al. 1993. J. Clin. Microbiol. vol. 31, pp. 3063-3067; Begum et al. 1993. J. Clin. Microbiol. vol. 31, pp. 3153-3156; Hull et al. 1993. vol. 31, pp. 1167-1172), for EHEC having the eaeA gene (Gannon et al. 1993. J. Clin. Microbiol. vol. 31, pp. 1268-1274) and for the large 60-MDa plasmid (Levine et al. 1987. J. Infect. Dis. vol. 156, pp. 175-182). These methods suffer from the drawbacks mentioned above, however, and the search for an improved method has continued in an effort to provide a rapid and sensitive assay for the detection of the microorganism.